Nosocomial infections by multiple antibiotic resistant bacteria are especially difficult to cure, pose a significant health risk and place an enormous burden on the economy. A leading cause of such nosocomial infections is the biofilm-forming Pseudomonas aeruginosa that primarily infects immune compromised individuals and those with severe burn wounds. In spite of P. aeruginosa being among the leading cause of nosocomial infections, little is known about the in vivo biofilm phenotype and the bacterial factors that prevent wound healing and promote persistence of P. aeruginosa at the infection site. We recently discovered that P. aeruginosa biofilms display a profoundly different phenotype compared to their planktonic counterparts with more than 800 proteins and 2000 genes being differentially expressed in biofilms. Among the virulence traits that defined the in vitro biofilm phenotype were ExoS-T, ExoA, LasB, and OprF-I that have recently been correlated with wound infections. Using a porcine burn wound model, we also demonstrated that bacteria formed matrix-encased, antimicrobial resistant microcolonies. The formation of P. aeruginosa biofilms in wounds correlated with a change in protein expression in P. aeruginosa-infected wounds as compared to non-infected wounds. Interestingly, the protein expression analysis also revealed an increased expression of proteins in in vivo P. aeruginosa biofilms that were found to be repressed in biofilms in vitro. In this R21 proposal, we intend to challenge current biofilm models by using emerging technologies and bold and aggressive approaches to investigate the role of P. aeruginosa biofilms in burn wound infections and to determine the P. aeruginosa in vivo biofilm phenotype. To our knowledge, this is the first attempt to tackle such a complex task. Our findings indicate the presence of novel and more virulent in vivo biofilm phenotypes in wounds. We hypothesize that biofilm formation correlates with clinical signs of infections and that in vivo P. aeruginosa biofilms display a new and more virulent phenotype than in vitro biofilms. To test our hypotheses, we propose to characterize the temporal formation of matrix-encased, antimicrobial resistant P. aeruginosa biofilms using an in vivo infection-biofilm model and to correlate our findings with signs of wound infections. We also propose to characterize the in vivo P. aeruginosa biofilm phenotype that is only displayed when normal microflora, and immunological and inflammatory cells are present. We anticipate that our findings will impact current treatment strategies to eradicate Pseudomonas biofilm infections by improving our understanding of P. aeruginosa pathogenesis and by identifying virulence factors that may be targeted for therapeutic intervention.